Capillary Electrophoresis Platform Research Articles

Joint application of A-InDels and miniSTRs for forensic personal, full and half sibling identifications, and genetic differentiation analyses in two populations from China

BackgroundPreviously, a novel multiplex system of 64 loci was constructed based on capillary electrophoresis platform, including 59 autosomal insertion/deletions (A-InDels), two Y-chromosome InDels, two mini short tandem repeats (miniSTRs), and an Amelogenin gene. The aim of this study is to evaluate the efficiencies of this multiplex system for individual identification, paternity testing and biogeographic ancestry inference in Chinese Hezhou Han (CHH) and Hubei Tujia (CTH) groups, providing valuable insights for forensic anthropology and population genetics research.ResultsThe cumulative values of power of discrimination (CDP) and probability of exclusion (CPE) for the 59 A-InDels and two miniSTRs were 0.99999999999999999999999999754, 0.99999905; and 0.99999999999999999999999999998, 0.99999898 in CTH and CHH groups, respectively. When the likelihood ratio thresholds were set to 1 or 10, more than 95% of the full sibling pairs could be identified from unrelated individual pairs, and the false positive rates were less than 1.2% in both CTH and CHH groups. Biogeographic ancestry inference models based on 35 populations were constructed with three algorithms: random forest, adaptive boosting and extreme gradient boosting, and then 10-fold cross-validation analyses were applied to test these three models with the average accuracies of 86.59%, 84.22% and 87.80%, respectively. In addition, we also investigated the genetic relationships between the two studied groups with 33 reference populations using population statistical methods of FST, DA, phylogenetic tree, PCA, STRUCTURE and TreeMix analyses. The present results showed that compared to other continental populations, the CTH and CHH groups had closer genetic affinities to East Asian populations.ConclusionsThis novel multiplex system has high CDP and CPE in CTH and CHH groups, which can be used as a powerful tool for individual identification and paternity testing. According to various genetic analysis methods, the genetic structures of CTH and CHH groups are relatively similar to the reference East Asian populations.

A dual-reference study design for understanding and improving AAV genome size analysis.

Recombinant adeno-associated virus (rAAV) is the leading platform of gene delivery for its long-lasting gene transformation and low immunogenicity. Characterization of the integrity and purity of the rAAV genome is critical to ensure clinical potency and safety. However, current rAAV genome characterization methods that can provide size assessment are either time-consuming or not easily accessible to general labs. Additionally, there is a lack of right reference standard for analyzing long single-stranded DNA (ssDNA) fragments. Here, we have developed an ssDNA assay on a microfluidic capillary electrophoresis platform using ssDNA reference standard. This assay provides size calling for ssDNA fragment, a detection sensitivity at ∼89pg/µL (3×1010 GC/mL AAV) for 5.1kb ssDNA fragment, and a turnaround time at ∼100s per sample with a high throughput sample analyzing capability. Moreover, we have observed that the annealing of AAV ssDNA subsequent to its release from the capsid might introduce an additional double-stranded DNA (dsDNA) peak. This phenomenon is dependent on the sample processing workflow. To avoid the risk of mischaracterization, we recommend the use of dual-reference standards in combination with other orthogonal methods to have a comprehensive understanding of the rAAV genome size and integrity.

A preliminary report on the exploration of salivary bacterial diversity by the multiplex SNaPshot assay

Salivary bacterial community composition is associated with the host’s internal and environmental factors, which have potential applications in forensic practice. The 16S rRNA gene sequencing is the most commonly used strategy for detecting salivary bacterial diversity; however, its platforms are not compatible with capillary electrophoresis (CE) platforms commonly used for forensic applications. Therefore, we attempted to detect the salivary bacterial diversity using a single nucleotide polymorphism (SNP) assay. Salivary bacterial diversity varies among diverse geographic locations, making it a potential supplementary biomarker for forensic geographic sourcing. To evaluate the performance of the multiplex SNaPshot assay, saliva samples from three geographic locations in China were analyzed using the multiplex SNaPshot assay and 16S rRNA gene sequencing. We screened SNPs from two high-relative-abundance salivary genera (Streptococcus and Veillonella) to construct a multiplex SNaPshot system that can be used on the CE platform. The stability and sensitivity of the multiplex SNaPshot system were also tested. A random forest classification model was used to classify samples from different regions to explore the ability of salivary bacteria to discriminate between geographic sources. Six bacterial SNPs were screened and a multiplex SNaPshot system was constructed. The stability results showed that the typing of salivary stains that were placed indoors for different days was not affected in this study. Two-thirds of mocked salivary stain samples showed more than 90% of typing results obtained for salivary stain samples with an input of 0.1 µl saliva. The results of principal coordinate analysis based on salivary bacterial diversity showed significant differences between samples from the three different geographic locations. The accuracy of the random forest classification was 66.67% based on the multiplex SNaPshot assay and 83.33% based on the 16S rRNA gene sequencing. In conclusion, this is the first attempt to detect salivary bacterial diversity using a multiplex SNaPshot bacterial SNP assay. The geographic difference in human salivary bacterial community composition was significant, as revealed by the multiplex SNaPshot assay; however, its performance in discriminating geographic sources was lower than that of 16S rRNA gene sequencing. This strategy based on bacterial SNP loci may favor the detection of human bacterial diversity in common forensic laboratories but requires further exploration in larger sample sizes and more bacterial SNP loci.

Systematical explorations of forensic feature and population genetic diversity of the Chinese Mongolian group from northwest China via a self-constructed Multi-InDel panel.

This study aimed to investigate the genetic polymorphisms and population characteristics of Chinese Mongolian group from northwest China (NCM) through a self-developed panel including 43 autosomal insertion/deletion (A-InDel) polymorphism genetic markers. Herein, 288 unrelated healthy individuals from the NCM group were employed to obtain the genetic data of 43 A-InDels through multiplex PCR amplification and InDel genotyping using capillary electrophoresis platform. In addition, multiplex population genetic analyses were performed between the NCM group and 27 reference populations. There were no deviations at 43 loci from Hardy-Weinberg equilibrium in the NCM group. The observed heterozygosity (Ho) values ranged from 0.312 8 to 0.559 2, and the combined power of discrimination (CPD) and cumulative probability of exclusion (CPE) values in the NCM group were 0.999 999 999 999 999 998 77 and 0.999 814, respectively. The forensic parameter values indicated that this panel was polymorphic and informative in the NCM group and could be used as an effective tool for forensic personal identification. Furthermore, the results of pairwise genetic distances, principal component analysis, multidimensional scaling analysis, phylogenetic tree construction, and admixture analysis among the NCM group and 27 reference populations revealed that there were closer genetic relationships between the NCM group and East Asian populations, especially Chinese Hui group (CHH) from the northwest China, which is consistent with the geographical location. These present findings contributed to the ongoing genetic explorations and insights into the genetic architecture of the NCM group.

Developmental validation of a custom-designed Multi-InDel panel: A five-dye multiplex amplification system for challenging DNA samples.

The continual investigation of novel genetic markers has yielded promising solutions for addressing the challenges encountered in forensic DNA analysis. In this study, we have introduced a custom-designed panel capable of simultaneously amplifying 41 novel Multi-insertion/deletion (Multi-InDel) markers and an amelogenin locus using the capillary electrophoresis platform. Through a developmental validation study conducted in accordance with guidelines recommended by the Scientific Working Group on DNA Analysis Methods, we demonstrated that the new Multi-InDel system exhibited the sensitivity to produce reliable genotyping profiles with as little as 62.5 pgof template DNA. Accurate and complete genotyping profiles could be obtained even in the presence of specific concentrations of PCR inhibitors. Furthermore, the maximum amplicon size for this system was limited to under 220 bp in the genotyping profile, resulting in its superior efficiency compared to commercially available short tandem repeat kits for both naturally and artificially degraded samples. In the context of mixed DNA analysis, the Multi-InDel system was proved informative in the identification of two-person DNA mixture, even when the template DNA of the minor contributor was as low as 50 pg. In conclusion, a series of performance evaluation studieshave provided compelling evidence that the new Multi-InDel system holds promise as a valuable tool for forensic DNA analysis.

Analysis of homozygous allele mismatches in paternity tests with massively parallel sequencing.

In paternity testing, short tandem repeats (STRs) allele mismatches are often detected. Nowadays, polymerase chain reaction- and capillary electrophoresis (CE)-based STR genotyping is the most commonly used method to distinguish alleles based on their length. However, it could not detect alleles of the same size with sequence differences. Massively parallel sequencing (MPS) can determine not only allele sizes but also sequences, which could explain the causes of allele mismatches. Additionally, more types of genetic markers can be detected in a single assay, which increases the discriminatory power and facilitates the analysis of paternity tests. In this study, we analyzed 11 cases with homozygous allele mismatches from routine DNA trio paternity tests using the CE platform. Samples were sequenced using the ForenSeq DNA Signature Prep Kit and the MiSeq FGx Sequencing System. The results show that of the eight father-child mismatch cases and three mother-child mismatch cases, five cases with D5S818 and D8S1179 and one case at D13S317 were classified as non-amplification. The other three cases and two cases could be defined as mutations. This study suggests that MPS-based STR genotyping can provide additional information that allows more accurate interpretation of allelic mismatches in paternity testing.

Development of a novel panel for blood identification based on blood-specific CpG-linked SNP markers.

Blood-containing mixtures often appear in murder and robbery cases, and their identification plays a significant role in solving crimes. In recent years, the co-detection of DNA methylation markers (CpG) and single nucleotide polymorphism (SNP) markers has been shown to be a promising tool for the identification of semen and its donor. However, similar research on blood stains that are frequently found at crime scenes has not yet been reported. In this study, we employed blood-specific CpG-linked SNP markers (CpG-SNP) for blood-specific genotyping and the linking of blood and its donor. The tissue-specific CpG markers were screened from the literature and further verified by combining bisulfite conversion with amplification-refractory mutation system (ARMS) technology. Meanwhile, adjacent SNP markers with a minor allele frequency (MAF) greater than 0.1 were selected within 400 bp upstream and downstream of the CpG markers. SNP genotyping was performed using SNaPshot technology on a capillary electrophoresis (CE) platform. Finally, a multiplex panel, including 19 blood-specific CpG linked to 23 SNP markers, as well as 1 semen-specific CpG, 1 vaginal secretion-specific CpG, and 1 saliva-specific CpG marker, was constructed successfully. The panel showed good tissue specificity and blood stains stored at room temperature for up to nine months and moderately degraded (4 < DI < 10) could be effectively identified. Moreover, it could also be detected when blood content in the mixed stains was as low as 1%. In addition, 15 ng of DNA used for bisulfite conversion was required for obtaining a complete profile. The cumulative discrimination power of the panel among the Han population of northern China could reach 0.999983. This is the first investigation conducted for the simultaneous identification of blood and its donor regardless of other body fluids included in mixed stains. The successful construction of the panel will play a vital role in the comprehensive analysis of blood-containing mixtures in forensic practice.

Development and validation of a custom panel including 114 InDels using massively parallel sequencing for forensic application.

Insertion/deletion polymorphisms (InDels) have particular characteristics, such as a relatively low mutation rate, small amplicon size, and no stutter artifacts when genotyped via the capillary electrophoresisplatform. It would be an important complementary tool for individual identification and certain kinship analyses. At present, massively parallel sequencing (MPS) has shown excellent application value in forensic studies. Therefore, in this study, we developed a custom MPS InDel panel that contains 114 InDels [77 autosomal InDels (A-InDels), 32 X-chromosomal InDels (X-InDels), and 5 Y-chromosomal InDels) based on previous studies. To assess this panel's performance, several validation experiments were performed, including sensitivity, inhibitor, degraded DNA testing, species specificity, concordance, repeatability, case-type samples, and population studies. The results showed that the lowest DNA input was 0.25ng. All genotypes were obtained in the presence of 80ng/µL humic acid, 2000µmol/L calcium, 3000µmol/L EDTA and indigo. In degraded DNA testing, 90% of loci could be detected for 16-day-old formalin-fixed hearts. In addition, this panel has good species specificity. The values of combined power of discrimination and the combined power of exclusion for 77 A-InDels were 1-3.9951×10-32 and 1-4.2956×10-7 , respectively. The combined mean exclusion chance for 32 X-InDels was 0.99999 in trios and 0.99904 in duos. The validation results indicate that this newly developed MPS multiplex system is a robust tool for forensic applications.

Internal validation study to assess the SeqStudio™ for human identification’s performance

The SeqStudio™ for human identification (HID) is a new benchtop capillary electrophoresis (CE) platform recently developed by Applied Biosystems for genotyping and sequencing short tandem repeat (STR) fragments. Compared to the previous series of CE systems developed by this maker, it is more compact and easier to use. Moreover, by allowing the detection of 4 to 8 fluorescent dyes, it seems to be fully compatible with the different kits of autosomal and gonosomal STR markers usually used in forensic genetics, which are available in trade and supplied by various manufacturers. However, being a new CE model, before its routine use in forensic genetics applications, it should undergo appropriate analytical validation studies in its own laboratories to understand its potential and limitations. A series of experiments on DNA samples coming from cell line controls, using the GlobalFiler™ IQC Amplification Kit, were carried out to meet this purpose. The SeqStudio™ Genetic Analyzer for HID’s findings on genotyping reproducibility (precision and accuracy of sizing), sensitivity, signal variability between dyes (intra- and inter-color channel balance), and stutter ratios are reported. These findings confirm the validity of this new CE system and its capability to generate reliable results.

Applying amplification refractory mutation system technique to detecting cell-free fetal DNA for single-gene disorders purpose.

Non-invasive prenatal diagnosis for single-gene disorders (NIPD) is still in development and deserves further study. The advent of next-generation sequencing technology significantly improved the detection of multiple mutations for non-invasive prenatal diagnosis for single-gene disorder purposes. However, bespoke amplicon-based NGS assays are costly. In this study, we developed a new strategy for non-invasive prenatal screening for single-gene disorders based on a capillary electrophoresis (CE) platform using an amplification refractory mutation system (ARMS)-PCR technique. Allele-specific primers for several disease-correlated mutations were designed, and subsequently, sensitivity and specificity assays were conducted. Assays on simulated two-person DNA mixtures showed that three primers targeting the mutant allele could detect minor DNA components in 1:500 mixtures. All primers showed positive results at 0.01ng of the template DNA. Cell-free fetal DNA was extracted from a pregnant woman's peripheral blood for the detection of paternally inherited mutations. Our results showed that one primer successfully amplified the mutant allele of fetal DNA in maternal plasma, which was confirmed by genotyping the genomic DNA extracted from amniotic fluid. This study suggested that the ARMS-PCR technique, a fast and cost-effective method, might be a promising method used to target de novo or paternally inherited pathogenic mutations in maternal plasma.

Forensic Validation Studies of a Novel 35-InDel Multiplex Polymerase Chain Reaction System

Abstract Background: A difficulty associated with forensic applications is the detection of degraded biological materials. Due to the large amplicon sizes of short tandem repeat alleles, valid genotyping results cannot be obtained from degraded biological materials. Recently, insertion/deletion (InDel) polymorphisms have been used in forensic applications for their widespread distributions in the human genome, short amplicon sizes, and low mutation rates. Purpose: Human identification InDel panels have mostly been designed for European populations. Therefore, our laboratory independently developed a multiplex polymerase chain reaction (PCR) system with 35 polymorphic InDel loci to be used for human identification in China. Forensic validation studies were conducted on this novel 35-InDel multiplex PCR system. Methods: The 35 InDel loci were screened in the database, and then used with the traditional PCR amplification and capillary electrophoresis platform combined with five-color fluorescence parallel detection technology. Validation studies were performed on this novel panel, including accuracy, repeatability and reproducibility, species specificity, sensitivity, stability, forensic case sample detection, and mixture studies. In addition, forensic efficiency assessments were conducted in populations from different continents. Results: The data of validated studies indicated that the novel 35-InDel panel was accurate, stable, and efficient for forensic purposes. For human identification, the cumulative power of discrimination values for the these 35 InDel loci in East Asian, South Asian, European, American, and African populations were 0.999999999999995, 0.999999999999995, 0.999999999999971, 0.9999999999999960, and 0.999999999998166, respectively. Conclusions: In this study, a set of 35 InDel loci were conducted in a multiplex amplification system for human identification of degraded DNA sample, and this new assay was efficient and stable. The present results suggested that the 35-InDel panel was a reliable tool for forensic use and could be efficiently used for human identification in the East Asian populations.

Performance evaluation of an in-house panel containing 59 autosomal InDels for forensic identification in Chinese Hui and Mongolian groups

In recent years, a novel multiplex system containing two mini-short tandem repeats, 59 autosomal InDels, two Y-chromosomal InDels, and the Amelogenin gene with all amplicons less than 200 bp has been constructed and validated by ourselves for forensic degration sample, and its forensic application efficiency has been studied in Chinese some populations. Herein, the population genetic polymorphisms of these loci were investigated in Chinese Hui (n = 249) and Mongolian (n = 222) ethnic groups using direct multiplex amplification and capillary electrophoresis platform. The forensic identification efficiencies of this self-developed system were further evaluated in these two groups. And the results showed that the values of the combined power of discrimination were 0.9999999999999999999999999999006 (Hui) and 0.999999999999999999999999999738 (Mongolian), respectively. Moreover, the combined power of exclusion values were 0.99999817 (Hui) and 0.99999779 (Mongolian). The 59 autosomal InDels used in this study exhibited high forensic identification efficiencies in 10 East Asian populations, which was also expected to be a new powerful tool for identifying degraded biological materials in East Asian populations.

Improving the system power of complex kinship analysis by combining multiple systems

Complex kinship analysis is a critical issue in forensic genetics. To address this issue, 55 STRs and 94 SNPs collected from four commercial forensic typing kits [three kits were based on a capillary electrophoresis (CE) platform and one was based on a next-generation sequencing (NGS) platform] were employed to test the system power for 2nd-degree and 3rd-degree kinship analysis. To measure the kinship index in related individuals, likelihood ratios (LRs) were calculated based on length and sequence polymorphism information (LRlength and LRsequence, respectively) from simulation as well as true pedigree samples. LRs calculated based on sequence information are generally higher than those based on length information. The sensitivity, specificity, and effectiveness to distinguish the 2nd- and 3rd-degree kinship were estimated from four marker sets with different numbers of markers. As expected, system power for kinship analysis improved by increasing the number of markers and using LRsequence, instead of LRlength. Furthermore, the system power based on 55 STRs from the CE platform is equal to the 40 STRs and 94 SNPs from one CE kit and the kit based on NGS platform for both 2nd-degree and 3rd-degree kinship analysis. For discrimination of 2nd-degree kinship, the system effectiveness is 86.63% with an error ratio < 0.01 using the 55 STRs from the CE platform. Using sequence information from the 55 STRs and 94 SNPs, the system effectiveness is 94.43%, with an error ratio < 0.001 for 2nd-degree kinship analysis and 64.34% with an error ratio < 0.05 for 3rd-degree kinship analysis, indicating that these markers are powerful for 2nd-degree kinship analysis and can be used for 3rd-degree kinship analysis.

Typing of semen-containing mixtures using ARMS-based semen-specific CpG-InDel/STR markers.

Mixed traces are common biological materials found at crime scenes, and their identification remains a significant challenge in the field of forensic genetics. In recent years, DNA methylation has been considered as a promising approach for body fluid identification, and length polymorphic loci are still the preferred markers for personal identification. In this study, we used tissue-specific CpG sites with linked insertion or deletion (InDel) or short tandem repeat (STR) markers (CpG-InDel/STR) for both body fluid and individual identification. The tissue-specific CpG loci, which were all selected from the previous reports, were analyzed using a combination of bisulfite conversion and amplification refractory mutation system-multiprimer-PCR technology. InDels or STRs, which were selected within 400bp upstream or downstream of the semen-specific CpG loci, were analyzed using a capillary electrophoresis platform. Eventually, we successfully constructed a panel containing 17 semen-specific CpG-InDel/STR compound markers compassing 21 InDels/STRs, 3 body-fluid positive controls (vaginal secretion-, saliva-, and blood-specific CpG), and 1 gender identification locus. Using this panel, full genotyping of individuals could be obtained successfully with 50ng DNA input. Semen stains stored at room temperature for 7months and degraded samples that were heat treated for up to 6h were still identified efficiently. For semen containing mixed stains, it is also useful when the semen content is as low as 3.03%. Moreover, the cumulative discrimination power of this panel is 0.9999998. In conclusion, it is a robust panel enabling the validation of both the tissue source and individual identification of semen containing mixed stains and can be employed as an alternative solution for forensic case investigation.

Forensic and genetic landscape explorations of Chinese Kyrgyz group based on autosomal SNPs, Y-chromosomal SNPs and STRs

To assess the effect of population genetic polymorphism on forensic research, we investigated the genetic polymorphisms of Chinese Kyrgyz group (n = 98) and evaluated forensic application values in Chinese Kyrgyz group and other 26 reference populations at 90 autosomal SNPs, and then combined with 34 SNPs and 37 STRs on Y chromosome to reveal the genetic background of Kyrgyz group in autosomal and Y-chromosomal inheritances, respectively. The 90 autosomal SNPs and 34 Y-chromosomal SNPs were sequenced base on next generation sequencing technology, and 37 Y-chromosomal STRs were analyzed by capillary electrophoresis platform. The results showed that cumulative power of discrimination and cumulative power of exclusion of 90 autosomal SNPs in the panel met the application need of forensic genetics in Kyrgyz group. The forensic effectivenesses of the panel were high in all 27 populations, although there were genetic differences among these populations. The forensic effectiveness of the panel was relatively higher in the European populations, but relatively lower in the African populations. The population genetic results indicated that the Kyrgyz group had the relatively closer genetic relationships with the reference East Asian populations at autosomal SNPs, and there were gene exchanges between the Kyrgyz group and East Asian, European populations based on the analytical results of autosomal SNPs, Y-chromosomal SNPs and STRs.

Application Progress of Massively Parallel Sequencing Technology in STR Genetic Marker Detection.

In recent years, more and more forensic genetics laboratories have begun to apply massively parallel sequencing (MPS) technology, that is, next-generation sequencing (NGS) technology, to detect common forensic genetic markers, including short tandem repeat (STR), single nucleotide polymorphism (SNP), the control region or whole genome of mitochondrial DNA (mtDNA), as well as messenger RNA (mRNA), etc., for forensic practice, such as individual identification, kinship analysis, ancestry inference and body fluid identification. As the most widely used genetic marker in forensic genetics, STR is currently mainly detected by capillary electrophoresis (CE) platform. Compared with CE platform, MPS technology has the advantages of simultaneous detection of a large number of genetic markers, massively parallel detection of samples, the polymorphism of sequence detected by NGS makes STR have the advantages of higher resolution and system efficiency. However, MPS technology is expensive, there is no uniform standard so far, and there are problems such as how to integrate MPS-STR data with the existing CE-STR database. This review summarizes the current status of the application of MPS technology in the detection of STR genetic markers in forensic genetics, puts forward the main problems that need to be solved urgently, and prospects the application prospect of this technology in forensic genetics.

Development and Performance Evaluation of a Novel Ancestry Informative DIP Panel for Continental Origin Inference.

Ancestry informative markers (AIMs) are useful to infer individual biogeographical ancestry and to estimate admixture proportions of admixed populations or individuals. Although a growing number of AIM panels for forensic ancestry origin analyses were developed, they may not efficiently infer the ancestry origins of most populations in China. In this study, a set of 52 ancestry informative deletion/insertion polymorphisms (AIDIPs) were selected with the aim of effectively differentiate continental and partial Chinese populations. All of the selected markers were successfully incorporated into a single multiplex PCR panel, which could be conveniently and efficiently detected on capillary electrophoresis platforms. Genetic distributions of the same 50 AIDIPs in different continental populations revealed that most loci showed high genetic differentiations between East Asian populations and other continental populations. Population genetic analyses of different continental populations indicated that these 50 AIDIPs could clearly discriminate East Asian, European, and African populations. In addition, the 52 AIDIPs also exhibited relatively high cumulative discrimination power in the Eastern Han population, which could be used as a supplementary tool for forensic investigation. Furthermore, the Eastern Han population showed close genetic relationships with East Asian populations and high ancestral components from East Asian populations. In the future, we need to investigate genetic distributions of these 52 AIDIPs in Chinese Han populations in different regions and other ethnic groups, and further evaluate the power of these loci to differentiate different Chinese populations.

Set of 15 SNP-SNP Markers for Detection of Unbalanced Degraded DNA Mixtures and Noninvasive Prenatal Paternity Testing.

Unbalanced and degraded mixtures (UDM) are very common in forensic DNA analysis. For example, DNA signals from criminal suspects are masked by a large amount of DNA from victims, or cell-free fetal DNA (cffDNA) in maternal plasma is masked by a high background of maternal DNA. Currently, detecting minor DNA in these mixtures is complex and challenging. We developed a new set of SNP-SNP microhaplotypes with short amplicons, and we successfully genotyped them using the new method of amplification-refractory mutation system PCR (ARMS-PCR) combined with SNaPshot technology based on a capillary electrophoresis (CE) platform. This panel reflects a high polymorphism in the Southwest Chinese Han population and thus has excellent potential for mixture studies. We evaluated the feasibility of this panel for UDM detection and noninvasive prenatal paternity testing (NIPPT). Fifteen SNP-SNPs detected minor DNA of homemade DNA mixtures, with a sensitivity of 0.025–0.05 ng and a specificity of 1:1,000. In addition, the panel successfully genotyped degraded DNA from single and mixed samples. Finally, 15 SNP-SNPs were applied to 26 trios. All samples displayed positive results with at least one marker to detect cffDNA. Besides, all fetal alleles in maternal plasma were confirmed by genotyping fetal genomic DNA from amniocentesis and paternal genomic DNA from peripheral blood. The results indicated that the SNP-SNP strategy based on the CE platform was useful for UDM detection and NIPPT.

Dual detection high-speed capillary electrophoresis for simultaneous serum protein analysis and immunoassays

Serum protein electrophoresis (SPE) separates serum proteins into bands whose shape and amplitude can alert clinicians to a range of disorders. This is followed by more specific immunoassays to quantify important antigens and confirm a diagnosis. Here we develop a high-speed capillary electrophoresis (HSCE) platform capable of simultaneous SPE and immunoassay measurements. A single laser excitation source is focused into the detection zone of the capillary to measure both refractive index (SPE) and fluorescence signals (immunoassays). The refractive index signal measures characteristic SPE profiles for human serum separated in 100 mM boric acid (pH 10), 100 mM arginine (pH 11), and 20 mM CHES (pH 10). For the immunoassay, the fluorescence electropherograms reveal that CHES provides the optimal buffer for measuring the immunocomplex and separating it from the free antigen. Immunoassays in CHES yield a LOD of 23 nM and a LOQ of 70 nM for the detection of fluorescein. The high pH reduces protein adsorption but reduces antibody affinity. Preliminary studies carried out in 50 mM barbital at pH 8 show improved stability of the immunocomplex and better separation for immunoassay quantification. Further optimization will open new capabilities for measuring orthogonal diagnostic signals in seconds with HSCE.

Genetic characterization evaluation of a novel multiple system containing 57 deletion/insertion polymorphic loci with short amplicons in Hunan Han population and its intercontinental populations analyses.

Deletion/insertion polymorphism (DIP) is a promising genetic marker of DNA length polymorphism. However, there are relatively few studies on the exploration of DIP genetic polymorphisms and investigation of population genetic data at present, which limits its application in forensic identification. In this study, the genetic polymorphisms of 57 autosomal DIPs and forensic application evaluations of the novel panel were analyzed in Chinese Hunan Han population using capillary electrophoresis platform, andthe differences of genetic polymorphic distributions at these loci were compared among the Hunan Han and 26 reference populations. The present results showed the combination of total 57 DIPs could be a robust tool for forensic individual identification and paternity testing. Due to the different allele frequency distributions in the different continental populations, the system could also effectively distinguish among East Asian, European and African populations.